Diabetic model

ABSTRACT

The invention relates to an animal model for diabetes and a method for obtaining said animal model. The invention also relates to the uses of the animal model for screening for or testing compounds for treating or preventing diabetes symptoms. The invention further relates to an assay for determining an individual&#39;s susceptibility to developing diabetes. The invention also relates to nucleic acid molecules isolated from Ljungan virus and to polypeptides encoded by any portion of said nucleic acid molecule.

This application is a national stage application (35 U.S.C. § 371) ofInternational application number PCT/IB02/03957, filed Aug. 21, 2002,and claims priority under 35 U.S.C. § 119 from United Kingdomapplication number 0120437.9, filed Aug. 22, 2001, incorporated hereinin its entirety by reference.

TECHNICAL FIELD OF INVENTION

The present invention relates to a method for obtaining an animal modelfor diabetes. The present invention also relates to the uses of theanimal model for screening for or testing compounds which affectdiabetic symptoms. The present invention also relates to an assay fordetermining an individual's susceptibility to developing diabetes.

BACKGROUND OF THE INVENTION

Diabetes is a disease in which the body does not produce or use insulincorrectly. Insulin is a hormone that is required to convert sugar,starches and other food into energy needed for daily life. Insulin isproduced by the beta cells in the islets of Langerhans in the pancreas.Partial or total loss of these cells will result in partial or totalloss of insulin production.

There are two major types of diabetes.

Type 1 diabetes is an autoimmune disease in which the body actuallyfails to produce any insulin. Type 1 disease most often occurs inchildren and young adults but can develop at any age. Type 1 diabetes ischaracterized by total loss of beta cells so that the patient requiresinsulin by injection. Type 1 diabetes accounts for 10-15% of alldiabetes. Type 1 diabetes is strongly associated with auto-antibodiesand this association has become part of the definition/classification oftype 1 diabetes. Type 1 diabetes is discussed in greater detail below.

Type 2 diabetes is a metabolic disorder resulting from the body'sinability to make enough, or properly use, insulin. It is the mostcommon form of the disease. Type 2 diabetes accounts for 85-90% ofdiabetes.

The definitions of type 1 and 2 diabetes, however, are changing slowly.Auto-antibodies are found in type 2 diabetes patients and type 2diabetes is found in increasing numbers in children. As a result, thetraditional view of type 1 and 2 diabetes as two different diseases bothresulting in increased blood glucose levels is shifting to the view thatthere is a large grey zone with patients in between the two extremes.This view is important when evaluating the usefulness of differentanimal models.

Both genetic and environmental factors are believed to be involved inthe development of type 1 (insulin dependent) diabetes (for reviews seeLeslie et al., Diabetologia, 42, 3-14, 1999; and Schranz et al., Diab.Metab. Rev., 14, 3-29, 1998). The HLA Class II region is the strongestgenetic component, but other genes and loci have been implicated ascontributing to a genetic predisposition to the disease (reviewed inSchranz et al., 1998 (supra)). Monozygotic twin studies show only 20-30%concordance of type 1 diabetes indicating a significant contribution ofenvironmental factors (Kyvik et al., BMJ, 311, 913-7, 1995). The role ofenvironmental factors is also supported by the fact that more than 85%of new onset patients do not have a first degree relative with thedisease (Dahlquist et al., Diabetologia, 32, 2-6, 1989).

Worldwide, there is a large variation in the incidence of type 1diabetes, ranging from more than 40 patients per 100,000 in Finland to1-2 cases per 100,000 in Japan (Onkamo et al., Diabetologia, 42,1395-403, 1999). Seasonal variation in incidence rate, together withserological studies, have suggested viral infections as a majorenvironmental risk factor for type 1 diabetes (for reviews see Jun etal., Diabetologia, 44, 271-285, 2001; Rayfield et al., Diab./Metab.Rev., 3, 925-57, 1987; and Vaarala et al., Diabetes Nutr. Metab., 12.,75-85, 1999). Congenital rubella virus infection (Menser et al., Lancet,i, 57-60, 1978) or different members in the enterovirus genus are mostoften implicated as an etiologic agents in diabetes development (Yoon,Do Viruses Play a Role in the Development of Insulin-dependentDiabetes?, 1991; Vaarala et al., 1999, (supra)). Signs of enterovirusinfection during pregnancy (Dahlquist et al., Diabetologia, 32, 2-6,1989; and Hyoty et al, Diabetes, 44, 652-657, 1995) and in some infantswho developed islet cell autoantibodies and later type 1 diabetes(Lonnrot et al., Diabetes, 49, 1314-8, 2000) further supports thishypothesis. Both Coxsackie B and rota virus contain peptide sequencesalso found in the islet autoantigens glutamate decarboxylase (GAD65)(Kaufman et al., J. Clin. Invest., 89, 283, 292, 1992), thetyrosine-phosphatase like protein IA-2 (Honeyman et al., Diabetes, 49,1319-1324, 2000) or proinsulin (Rudy et al., Mol. Med., 1, 625-33, 1995)suggesting that T lymphocytes recognizing viral antigens may potentiallycontribute to islet autoimmunity by cross-reactivity or molecularmimicry. Indeed, cross-reactive GAD65 and rubella virus peptides wererecognized by T cells in type 1 diabetes patients (Ou et al.,Diabetologia, 43, 750-62, 2000). Since T cell tests that predict type 1diabetes are not yet available, standardized tests for GAD65, IA-2 orinsulin autoantibodies are useful markers to predict type 1 diabetes(for a review see Gottleib et al., Arum. Rev. Med., 49, 391-405, 1998).Rota virus seroconversion was reported to be associated with increasesin autoantibodies to GAD65, IA-2, and insulin suggesting that this virusinfection may trigger or exacerbate islet autoimmunity in geneticallysusceptible children (Honeyman et al., 2000 (supra)). Coxsackievirus-induced diabetes in mice was also associated with the developmentof GAD antibodies (Gerling et al., Autoimmunity, 6 49-56,1991). It isstill controversial, however, whether viruses cause beta celldestruction directly by a cytolytic infection in the islets orindirectly by initiating autoimmunity (Vreugdenhil et al., Clin. Infect.Dis., 31, 1025-31, 2000; and Kukreja et al., Cell Mol. Life Sci., 57,534-41, 2000).

Rodents are well-known reservoirs and vectors for viruses causingdisease in humans. Puumala virus causing Nephropathia Epidemica(Myhrman, Nordisk Medicinsk Tidskrift, 7, 739-794, 1934; and Niklassonet al., Lancet, 1, 1012-3, 1984) is one example of an important humanpathogen carried by bank voles. It has been demonstrated that theincidence rate of human Nephropathia Epidemica correlates with the volepopulation density during the previous year (Niklasson et al., Am. J.Trop. Med. Hyg., 53, 134-40, 1995). More recently, statistical evidencesuggests that type 1 diabetes in humans also tracks the 3- to 4-yearpopulation density cycles of the bank vole with a similar time lag(Niklasson et al., Emerg. Infect. Dis., 4, 187-93, 1998). It also wasobserved that a high frequency of bank voles trapped in the wild andkept in the laboratory for studies of stereotypic behavior (Schoeneckeret al., Appl. Anim. Behav. Sci., 68. 349-357, 2000) develop symptoms oftype 1 diabetes, i.e., polydipsia and glucosuria, at a high frequency.

Currently there are two main animal models of diabetes: the NOD (nonobese diabetic) mouse and the BB (bio breeding) rat. Both models involveanimals with insulin dependent diabetes. Both of the current models,however, fail to display important symptoms of human diabetes. The NODmouse, for example, shows gender preferences that are opposite to thehuman disease (i.e., more females than males develop the disease),develops mild diabetes, requires a long time before developingketoacidosis, and fails to develop autoantibodies to GAD65, 1A-2 orinsulin. The disease is genetically controlled in the NOD mouse and thecleaner the animal, the higher the frequency of diabetes.

The BB rat is also no ideal. The animals have lymphopenia controlled byan autosomal mutation on chromosome 4 and the development ofautoantibodies in inbred and specific pathogen free BB rats appearsnegligible. None of these BB rats develop diabetes in association withan infectious agent.

Thus, there is a need to develop an improved method for obtaining ananimal model which displays the features of diabetes for both researchand therapeutic purposes.

SUMMARY OF THE INVENTION

The invention provides an animal model for human diabetes and methodsfor producing it. The invention also provides methods for screening foror testing compounds which affect diabetic symptoms comprising use ofthe animal model. The invention further provides an assay fordetermining an individual's susceptibility to developing diabetes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the histology of the pancreas in bank voles withoutdiabetes (panels a and b as well as e and f) and with diabetes (panels cand d as well as g and h). The histology of non-diabetic bank volesdemonstrates well-defined islets of Langerhans surrounded by aconspicuous and delicate capsule. Diabetic bank voles have dramaticislet cytopathology characterized by distinct vacuoles or fattyinfiltration of the pancreatic islets. Hematoxylin and Eosin stainingare shown in panels a-d while immunostaining for insulin and glucagonare shown in panels e-h. The immunostained sections demonstrate that thecytopathology affected only insulin positive cells, which were lostresulting in a redistribution of glucagon immunoreactive cells. Sizebars are indicated in each panel.

FIG. 2 shows the histology of the pancreas of a diabetic bank voledemonstrating islet infiltration of mononuclear cells followingHematoxylin and Eosin staining.

FIG. 3 shows the histology of the pancreas in bank voles without (panelsa and b) and with diabetes (panels c and d) following immunostainingwith the mouse 87-012 or 145L antiserum against Ljungan virus. Thebinding of the mouse antiserum was revealed with red vector staining.The sections were double stained with glucagon antiserum revealed withalkaline phosphatase and tetrazolium blue. The non-diabetic voles didnot show binding of mouse anti-Ljungan virus antibodies while theimmunostaining against glucagon stained cells in the periphery of theislets (panel a). The islets in diabetic bank voles showed varyingdegree of vacuolization or fatty infiltration of the pancreatic islets(panels b, c and d). The edges of these lesions are stained indicatingthe presence of Ljungan virus antigen. The glucagon immunostainingshowed redistribution of cells that became more pronounced the greaterthe lesions.

FIG. 4 shows that the bank voles have autoantibodies against islet cellautoantigens and against Ljungan virus in vitro translated antigens.Autoantibodies to GAD65 (panel a), IA-2 (panel b), insulin (panel c) aswell as Ljungan virus in vitro translated antigens are shown as in-houserelative Units on a log scale (wherein a 1/25 dilution of standard serumis equal to 100 units/ml). Group A animals were caught and bled in thewild and only 4% had diabetes. Group B bank voles were captive and 33%of the animals shown had diabetes. The levels of GAD65 (p<0.0001), IA-2(p<0.0001) and insulin (p<0.03) autoantibodies were increased in Group Bcompared to Group A bank voles. The autoantibody levels of both GAD65and IA-2 were higher in diabetic as compared to non-diabetic Group Bbank voles as indicated in the Figure. The data in panel d demonstratethat antibodies to Ljungan virus in vitro translated antigens were alsoincreased in diabetic compared to non-diabetic bank voles. Data forindividual bank voles are shown.

FIG. 5 shows the sequence similarities and cross-reactivity betweenGAD65 autoantibodies and mouse or human anti-Ljungan virus antibodies.Sequence comparisons between the predicted amino acid sequence ofLjungan virus (serotype 87-012) and type 1 diabetes associatedautoantigens are shown in panel a. The data compares the 87-012 Ljunganvirus sequence and regions of potential molecular mimicry to GAD65, IA-2and insulin. Areas of homology are boxed, with identical amino acidsindicated by a dot, similar amino acids are boxed, and non-similar aminoacids are plain type. Antibodies against Ljungan virus raised in mice(antiserum 87-012) showed cross-reactivity with human GAD65 (panel b).Radiobinding analysis to the 87-012 antiserum showedconcentration-dependent binding of ³⁵S-labeled Ljungan virus in vitrotranslated antigen (x-x) and human (o-o) but not mouse (•-•) GAJD65. Thecompetition at half maximal binding of the 87-012 antiserum betweenbinding of ³⁵S-labelled human GAD65 and unlabelled Ljungan virus antigen(x-x), human GAD65 (o-o) or human proinsulin (▾-▾) (panel c)demonstrates displacement by unlabelled Ljungan virus in vitrotranslated antigens as well as by recombinant human GAD65. FIG. 5 d.shows binding of different ³⁵S-labeled antigens including Ljungan virusantigen (x-x), human GAD65 (o-o) or mouse GAD65 (•-•) to the type 1diabetes human serum #591 (panel d). Human and mouse GAD65 bind equallywell and there are also significant levels of antibodies detecting theLjungan virus in vitro translated antigens. Competition at half maximalbinding to human serum 591 of ³⁵S -labelled Ljungan virus antigen andcold Ljungan virus antigen (x-x), human GAD65 (o-o) or human proinsulin(▾-▾) (panel e) showed displacement of cold Ljungan virus in vitrotranslated antigens but not of cold GAD65 or proinsulin. Allradioactivity values in cpm are mean values±SEM for 3-5 experiments TheSEM bars are within the size of the symbols unless indicated.

FIG. 6 shows the results of tests on sera from children with new onsettype 1 diabetes indicating the presence of anti-Ljungan virusantibodies. Ljungan virus antibodies were determined in two independenttests by either indirect immunofluorescence of cells used to propagatethe virus or by the radioligand binding assay with Ljungan virus invitro translated antigens. The radioligand binding assay correlated tothe indirect immunofluorescence test (p<0.001 at 95 confidenceinterval).

FIG. 7 shows the nucleotide sequence of Ljungan virus 87-012.

FIG. 8 shows the nucleotide sequence of Ljungan virus 145SL.

FIG. 9 shows the nucleotide sequence of Ljungan virus 174F.

DETAILED DESCRIPTION OF THE INVENTION

According to a first embodiment of the present invention, there isprovided a method for obtaining an animal model for human diabetes,comprising obtaining a mammal that has been determined to be infectedwith Ljungan virus.

The mammal may be any mammal including rodents such as rats, mice,hamsters, guinea pigs, rabbits, bank voles and field voles; cattle suchas cows; cats; dogs; and non-human primates. Preferably the mammal is arodent, a cat or a dog, more preferably the mammal is a rodent, mostpreferably the mammal is a bank vole.

It has been found that bank voles having type 1 diabetes are allinfected with Ljungan virus and that the presence of Ljungan viruscauses or at least contributes to the development of type 1 diabetes.

The term “Ljungan virus” as used herein refers to any Ljunganpicornavirus as defined in International PCT patent application WO98/11133, the disclosure of which is incorporated herein by reference.Preferably the Ljungan virus is Ljungan virus 87-012, the nucleotidesequence of which is shown in FIG. 7; Ljungan virus 145SL, thenucleotide sequence of which is shown in FIG. 8; or Ljungan virus 174F,the nucleotide sequence of which is shown in FIG. 9.

The presence of Ljungan virus can be determined using any standardprocedure including, but not limited to, virus isolation, detection ofLjungan virus antigen by ELISA or immunohistochemistry using antibodymolecules having affinity for Ljungan virus or detection of Ljunganvirus specific RNA sequences using PCR or by a labeled nucleic acidprobe capable of specifically hybridizing to Ljungan virus nucleic acid.The presence of Ljungan virus also can be determined by detecting forthe presence of Ljungan virus antibodies using a suitable test. Suitabletechniques for determining the presence of Ljungan virus or anti-Ljunganvirus antibodies are described in the examples below.

Use of the mammal obtained by the method according to the firstembodiment of present invention as a model for human diabetes has anumber of advantages over the prior art animal models of diabetesincluding:

-   -   a pathology that includes total destruction of the beta cells        without affecting the surrounding pancreas tissue;    -   no or minor signs of inflammatory cells and only modest        insulitis; and    -   the presence of auto-antibodies used as markers for human type 1        diabetes (antibodies to GAD 65, IA-2 and insulin) in most bank        voles obtained by the method according to the first embodiment        of the present invention.

The fact that the mammal obtained using the method according to thefirst embodiment of the present invention has features that mimic thehuman disease means that it closely represents the human disease and istherefore a particularly useful model of diabetes.

Preferably, the method according to the first embodiment of the presentinvention also comprises determining whether the mammal has high bloodglucose levels that can be reduced by insulin and signs of ketoacidosis.

Preferably, the mammal is a bank vole. The bank vole may be any speciesof bank vole. Preferably the bank vole is Clethrionomys glareolus. Themammal can be male or female. The bank vole may be obtained from thewild or may be the progeny of a bank vole obtained from the wild. It ispreferred that the bank vole obtained from the wild is obtained fromDenmark, Sweden or Finland. Alternatively, the bank vole may be alaboratory bred bank vole.

The term “diabetes” as used herein means type 1 or type 2 diabetes ordiabetes having a combination of symptoms of both type 1 and type 2diabetes. The type of diabetes developed by the mammal will depend onthe type of mammal. For example bank voles infected with Ljungan virusdevelop type 1 diabetes, whereas cats and dogs can develop type 1 ortype 2 diabetes or diabetes having a combination of symptoms of bothtype 1 and type 2 diabetes. It is currently believed that diabetes inhumans is not always type 1 or type 2 diabetes but that diabetes canfall somewhere between the two defined types wherein the individual hassome symptoms of both type 1 and type 2 diabetes. The term “diabetes” asused herein refers to diabetes characterized by high blood glucoselevels that can be reduced by insulin and signs of ketoacidosis. Thepresence of auto-antibodies to at least one of GAD65, IA-2 and insulinis an additionally preferred characteristic of type 1 diabetes accordingto the present invention. Additional features of diabetes such ashyperlipidemia, slowly progressive increase of hyperglycemia andvariable glucosuria as well as symptoms of hyperphagia and obesity alsomay be present in addition to the characteristics of type 1 diabetesdefined above in accordance with the situation in humans.

The term “high blood glucose levels” as used herein means blood glucoselevels that are at least 1.5 times as high, more preferably at least 3times as high and most preferably at least 5 times as high as the meanlevel of blood glucose found in the corresponding non-diabetic mammals.Non-diabetic mammals are mammals that do not show any symptoms ofdiabetes such as increased glucosuria. It is particularly preferred thata high blood glucose level is at least 150 mg/dl, more preferably atleast 200 mg/dl.

The term “reduced by insulin” as used herein means that the high bloodglucose levels can be reduced by the addition of insulin. Preferably theblood glucose levels can be reduced by about 30%, more preferably 60%and most preferably to approximately the level of a non-diabetic mammalsby the addition of insulin. As those skilled in the art will appreciate,the reduction in blood glucose levels will vary depending on the amountof insulin given to the mammal.

Signs of ketoacidosis include nausea, vomiting, stomach pain, deep andrapid breathing, flushed face, dry skin and mouth, fruity breath odor,rapid and weak pulse, low blood pressure. Ketoacidosis can be determinedby the measurement of keton bodies in the blood or in plasma or serum.

The method according to the first embodiment of the present inventionpreferably additionally comprises modulating the immune system of themammal to facilitate the development of diabetes. The modulation can beby suppressing or enhancing the immune system. The immune system of themammal can be modulated by any method including administratingimmunosuppressing or immunostimulating agents, altering the diet of themammal or subjecting the mammal to stress. Preferably the immune systemof the mammal is modulated by subjecting the mammal to stress. Themammal can be subjected to any form of stress that affects the immunesystem of the mammal including keeping the mammal in a cage. Inembodiments in which the mammal is a bank vole, it preferably is kept ina cage for at least 2 months, more preferably at least 3 months.Preferably the mammal is kept isolated in its own cage.

The present invention also provides the use a mammal infected withLjungan virus as a model of diabetes.

The mammal used as a model of diabetes is preferably obtained by themethod according to the first embodiment of the present invention.

The mammal can be used as a model of diabetes in order to investigatethe development and etiology of diabetes. The mammal can also be used totest candidate compounds for their effects on symptoms of diabetes. Inparticular, a candidate compound can be administered to the mammal andthe effects of the compound on symptoms of diabetes, such as bloodglucose levels, signs of ketoacidosis and glucosuria can be measured.

The mammal can also be used to screen for compounds having an effect onthe development of diabetes. Preferably the mammal is used to screen forcompounds that prevent the development of, or reduce the symptoms of,diabetes.

The present invention also relates to the use of compounds identified inthe above-described screening in the manufacture of a composition fortreating and/or preventing diabetes.

The present invention also relates to the use of cells obtained frommammals obtained by the method according to the first embodiment of thepresent invention. The cells can be used in a variety of in vitro assayswhich are well known to those skilled in the art.

In a second embodiment of the present invention there is provided amethod for producing diabetes in a mammal comprising infecting themammal with a Ljungan virus.

It has been found that mammals infected with Ljungan virus developdiabetes. The mammal infected with Ljungan virus can be used as a modelof diabetes whether or not symptoms of diabetes can be detected.

The mammal can be infected using any standard technique, including, butnot limited to, parenteral routes such as intravenous injection andintraperitoneal injection. Methods for determining the necessary viraldose leading to the development of diabetes can be easily determined bythose skilled in the art. In making such a determination, a number offactors are considered including the species of mammal, the rate ofviral replication, the route of infection, the age and sex of themammal. Preferably about 1,000 infection units are given to the mammal.

The mammal infected with Ljungan virus may develop type 1 or type 2diabetes or diabetes having a combination of symptoms of both type 1 andtype 2 diabetes.

The method according to the second embodiment of the present inventionpreferably additionally comprises modulating the immune system of themammal as described above with respect to the first embodiment of thepresent invention. Preferably the immune system of the mammal iscompromised by subjecting the mammal to stress as described above withrespect to the first embodiment of the present invention subsequent toinfection with the infectious agent.

By compromising the immune system of the mammal, it has been found thatthe animals develop diabetes more quickly. Without being bound to anyone theory, it is believed that the Ljungan virus can replicate at afaster rate leading to the development of diabetes in a shorter periodof time in immune-compromised animals.

The present invention also provides the use of a mammal infected with aLjungan virus as a model of diabetes. Preferably the mammal is obtainedby the method according to the second embodiment of the presentinvention.

The mammal infected with a Ljungan virus can be used as a model toinvestigate the development and etiology of diabetes. The mammal alsocan be used to test candidate compounds for their effects on diabetes.In particular, a candidate compound can be administered to a mammalinfected with a Ljungan virus and the effects of the compound onsymptoms of diabetes, such as blood glucose levels, signs ofketoacidosis, glucosuria, hyperlipidemia, a slowly progressive increasein hyperglycemia, symptoms of hyperphagia, obesity and insulinresistance, can be measured.

The mammal infected with a Ljungan virus can also be used to screen forcompounds having an effect on the development of diabetes. Preferablythe mammal is used to screen for compounds which prevent the developmentof, or reduce the symptoms of, diabetes.

The present invention also provides an assay for determining anindividual's susceptibility to developing diabetes comprising analyzinga sample from the individual in order to determine if the individual isinfected with a Ljungan virus, wherein infection with a Ljungan virusindicates a greater susceptibility to developing diabetes.

It has been found that children with an increased level of antibodiesagainst Ljungan virus have type 1 diabetes (i.e., serologically positivefor Ljungan virus infection). We have determined that a population ofchildren with diabetes has a much higher frequency of beingserologically positive for Ljungan virus than in a population of healthycontrol children.

The presence of Ljungan virus can be determined using any standardprocedure including immunohistochemistry using antibody molecules havingaffinity for Ljungan virus or by using a labeled nucleic acid probecapable of specifically hybridizing to Ljungan virus nucleic acid.Alternatively the presence of Ljungan virus can be determined bydetecting the presence of anti-Ljungan virus antibodies using a suitabletest. Suitable techniques for determining the presence of Ljungan virusor anti-Ljungan virus antibodies are described in the examples below.

The present invention also provides a method of treating an individualwho has developed diabetes or is susceptible to developing diabetescomprising administering an effective amount of a compound whichprevents or reduces Ljungan virus-induced diabetes.

Compounds which prevent or reduce the effects of Ljungan virus includeantibody molecules having affinity for Ljungan virus or any otheranti-viral agents. Methods for producing suitable antibody molecules arewell know to those skilled in the art.

The present invention also provides a method of vaccinating anindividual against a Ljungan virus infection, thereby preventing, atleast in part, the individual developing diabetes.

Vaccines of the invention may comprise any antigenic portion of theLjungan virus (e.g. a protein displayed on the surface of the virus) orby using an attenuated form of the Ljungan virus. Methods for producingvaccines based on antigenic components or attenuated forms of the virusare well known to those skilled in the art and are described in avariety of literature know to those skilled in the art (see TextbookField's Virology by David M. Knipe et al).

In another aspect, the invention includes nucleic acid moleculesisolated from Ljungan viruses or any portion thereof. In someembodiments, the nucleic acid is the one shown in any one of FIG. 7, 8or 9 or any portion thereof.

The invention further comprises a nucleic acid molecule encoding aLjungan virus polypeptide, or fragments thereof. In some embodiments,the nucleic acid is operably linked to one or more expression controlsequences. In some embodiments, the nucleic acid molecule or fragment isincorporated into a vector. In some embodiments, the vector is anexpression vector.

The invention also provides host cells comprising a nucleic acid orvector of the invention. The host cell can be prokaryotic or eukaryotic.The choice of host cells for expressing a Ljungan virus polypeptide iswell-known in the art.

The invention further provides methods for producing a Ljungan viruspolypeptide or fragment thereof comprising culturing host cells of theinvention under conditions suitable for the expression of thepolypeptide and recovering the polypeptide.

The invention further comprises a vaccine comprising at least oneLjungan virus polypeptide or an immunogenic fragment thereof. In someembodiments, the vaccine comprises a plurality of Ljungan viruspolypeptides or immunogenic fragments thereof. The Ljungan viruspolypeptides can be from the same or different strains of Ljungan virus.Vaccines comprising polypeptides from different strains are useful toprevent or inhibit infection by a broader range of Ljungan virus inconditions caused by Ljungan virus infection, including diabetes.

In some embodiments, Ljungan virus polypeptides are a component of amultivalent vaccine that comprises one or more components from otherpathogens, including human pathogens.

In some embodiments, a vaccine of the invention comprises an adjuvant.Methods for selecting an adjuvant for use in the vaccine are well-knownto those of skill in the art.

The invention further provides an antibody that specifically bindsLjungan virus or a Ljungan virus polypeptide. In some embodiments, theantibody specifically binds one or more of the Ljungan viruspolypeptides shown in FIGS. 7-9.

EXAMPLES

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purposes ofillustration only and are not to be construed as limiting the scope ofthe invention in any manner.

Materials and Methods

Wild caught bank voles (Group A). Group A bank voles represent 101animals from a single trapping session. These bank voles were tested atthe trap for glucosuria and then euthanized. Heart-blood samples forblood glucose, ketosis, lipids and antibody analyses were takenimmediately after the voles were killed. Blood samples were eitherimmediately analyzed for blood glucose and ketones or centrifuged for 25minutes at 1,000×g and plasma stored at −30° C. Pancreas was dissectedand fixed in 4% paraformaldehyde followed by ethanol before beingembedded in paraffin.

Voles caught in the wild and kept in the laboratory (Group B). In twoother trapping sessions, 163 voles were caught and transferred to thelaboratory as previously described (Schoenecker et al., Appl. Anim.Behav. Sci, 68, 339-347, 2000; Schoenecker et al., Appl. Anim. Behav.Sci., 68. 349-357, 2000). The animals were housed individually in smallbarren cages of transparent plastic (13.5×16.0×22.5 cm) under conditionsof minimum extraneous disturbance and with a twelve-hour light regime(8.00-20.00 h). The cages were supplied with a woodcutting bed, and food(standard rat chow) and water were available ad libitum. Cage cleaningand body weight measurements were performed once every week. A portionof grain mixture was given when the cages were cleaned. Diabetesdevelopment was followed by measurements of water intake, glucosuria,and blood glucose and ketonemia determined after bleeding from the retroorbital plexus. Polydipsic voles were characterized by >21 ml/day waterintake compared with non-polydipsic voles for which daily intake did notexceed 12 ml.

Histological analysis and immunocyto-chemistry. Standard hematoxylin andeosin staining was carried out on samples fixed in 4% paraformaldehyde,embedded in paraffin, cut into 5 micron sections, and affixed to slides.Sections were deparaffinized, rehydrated, and stained for three minutesin Gill's hematoxylin and for one minute in Eosin Y. Stained sectionswere dehydrated and mounted.

In the immunohistochemistry tests, pancreas fixed in 4% paraformaldehydeand embedded in paraffin were cut into 5 micron thick sections, affixedto slides, deparaffinized and rehydrated. The sections were blocked for30 min at RT in PBS containing 0.05% Tween 20 (Sigma, St. Louis, Mo.),1% BSA (Sigma), 2% normal horse (in the case of staining for Ljunganvirus antisera) or 2% normal goat (in the case of insulin or glucagonstaining) serum (Vector Laboratories, Burlingame, Calif.), and 4drops/ml Avidin solution (Avidin/Biotin blocking kit, VectorLaboratories). The primary antibody was diluted in PBS with 0.05% Tween20, 1% BSA, 2% normal serum, and 4 drops/ml Biotin solution (fromAvidin/Biotin blocking kit, Vector Labs) to 1:100 (guinea piganti-insulin and rabbit anti-glucagon, Zymed Laboratories, S. SanFrancisco, Calif.) or 1:500 (mouse Ljungan virus antiserum). Slides wereexposed to the primary antibody solution for 60 minutes at roomtemperature or overnight at 4° C. Slides were then washed in PBS,incubated for 30 minutes at room temperature with a biotinylatedsecondary antibody (goat anti-rabbit IgG, goat anti-guinea pig IgG, orhorse anti-mouse IgG (Vector Labs) diluted 1:500 in PBS, and washedagain. The slides were next incubated for 30 minutes at room temperaturein alkaline phosphatase streptavidin conjugate (Vector Labs) at a 1:200dilution, washed in PBS, and reacted with the Vector Red or VectorBCIP/NBT alkaline phosphatase substrate kit. Finally, slides werecounter stained with methyl green, dehydrated, and mounted. All slideswere coded and scored independently by two readers.

Immunfluorescence assay for Ljungan virus antibodies. Sera from childrenwith type 1 diabetes and controls were tested for presence of antibodiesto Ljungan virus using an indirect immunofluorescence test (IFT). Apreviously described IFT protocol (Niklasson et al., J. Infect. Dis.,155, 369-76, 1987) was used to test antibody titers. Briefly, spotslides were prepared by incubating virus in Green Monkey Kidney cellsfor 8-10 days. At signs of discrete cytopathic effects (CPE), cells wereremoved from the flask with a rubber policeman and applied ontomicroscope slides, air dried, fixed in cold (4° C.) acetone and storedat −70° C. until used. The titer was determined after incubating theserum, diluted in PBS, on the slides at 37° C. for 1 h in a moistchamber and bound antibodies were detected by incubating FITC-conjugatedgoat anti-human IgG (Sigma, St Louis, Mo.) for 1 h at 37° C. Patient andcontrol sera was first tested at a 1:8 dilution using three Ljunganvirus isolates (87-012, 145SL, 174F). Any sera scoring positive for anyof the three isolates were titrated again using all three isolatesseparately. Patients and controls positive to one or several isolates ata titer of 32 or higher was considered positive.

Radioligand binding assays for GAD65 and IA-2 antibodies. GAD65 and IA-2antibodies were analyzed as described (Grubin et al., Diabetologia, 37,344-350, 1994; Hampe et al., J. Clin. Endocrinol. Metab., 85, 4671-9,2000; Vandewalle et al., Diabetes Care, 20, 1547-1552, 1997). GAD65 andIA-2 antibody levels were expressed in U/ml for GAD65 and IA-2antibodies using the WHO/JDF standard (Mire-Sluis et al., Diabetologia,43, 1282-1292, 2000).

Insulin autoantibodies (IAA). IAA were measured using a method for smallplasma/serum samples (Williams et al., Journal of Autoimmunity, 10,473-478, 1997). An in-house serum sample was used as the standard toexpress the data in arbitrary U/ml. Recombinant human insulin (NovoNordisk, Copenhagen, Denmark) was used to determine IAA specificity asdescribed (Williams et al., (supra), 1997).

Radioligand binding assay for Ljungan virus antibodies. We used theLjungan virus cDNA (unpublished observation) in the coupled in vitrotranscription translation assay as described for GAD65 (Grubin et al.,(supra), 1994; Hampe et al., (supra), 2000). The Ljungan virus cDNA wastranslated into multiple components which were immunoprecipitated withLjungan virus mouse and guinea-pig antisera (data not shown) as well asfrom serum for both non-diabetic and diabetic bank voles and new onsettype 1 diabetic patients. The human 591 GAD65-positive serum (Mire-Sluiset al., (supra), 2000) showed high binding and was used as an in-housestandard to express antibody binding levels in arbitrary U/ml.

Competition experiments. Competition in binding between radioactive andcold antigens was carried out at half maximal binding of either theLjungan virus 87-012 mouse antiserum or the 591 human standard serumfound to be positive for antibodies against both Ljungan virus in vitrotranslated antigens and GAD65 (Mire-Sluis et al., (supra), 2000).Competition for binding of ³⁵S-labeled Ljungan virus in vitro translatedantigens was carried out with different concentrations of unlabeledLjungan virus in vitro translated antigens, recombinant human GAD65(DiamydAB, Stockholm, Sweden) or human proinsulin (Elli Lilly Company,Indianapolis, Ind.).

Type 1 diabetes patients and controls. A total of 53 children with amedian age of 10.1 years (range 2.3-16.4 years of age) were diagnosedwith type 1 diabetes at the St Göran Hospital and Astid Lindgren'sChildren's Hospital between 1992 and 1995. Within two days of diagnosis,blood samples were drawn for antibody analysis. Healthy children (7boys, median age 12.6 (7.8-16.8 years and 10 girls, median age 13.5(6.7-16.6 years ) were recruited from school classes in centralStockholm and children to personnel at the hospital. All children werepreviously healthy and without present medication. The Ethics Committeeat the Karolinska Institute, Stockholm, Sweden, approved the study.

Bioinformatics. To identify regions of high local homology between thevirus polyprotein and known diabetes autoantigens, we created a localdatabase of GAD65, IA-2 and insulin sequences and ran stand alone BLASTusing software from the NCBI (Altschul et al., Nucleic Acids Res., 25,3389-402, 1997). Alignments were compiled manually to align regions ofsimilarity onto the Ljungan protein sequence using CLUSTALW to determinesimilarity between non-homologous residues.

Statistics. The frequency of diabetes in the different groups wasanalyzed by Fischer exact test or Chi Square tests. Non-parametric testswere used to analyze differences in levels between groups. SpearmansRank Correlation was used to examine possible correlation betweendifferent parameters.

Example 1

Obtaining Bank Voles having Type 1 Diabetes

Development of Diabetes in Trapped Bank Voles

Bank voles were trapped from May to November in a forest habitat on theisland of Zealand, Denmark. In different continuous trapping sessions of30 days duration, 100 traditional live traps were set and inspectedtwice a day. Two groups of bank voles were analyzed for diabetes andpancreas histology in addition to type 1 diabetes associatedautoantibodies against insulin (Williams et al., (supra), 1997), GAD65(Grubin et al, (supra), 1994), and IA-2 (Lan et al., DNA and CellBiology, 13, 505-514, 1994) also known as ICA512 (Rabin et al., J.Immunol, 152, 3183-3187,1994) as well as antibodies against Ljunganvirus (Niklasson et al., Virology, 255, 86-93, 1999). Group A bank volesrepresents 101 trapped bank voles that were euthanized in the forest forimmediate examination of blood glucose, glucosuria, body weight,pancreas histology and antibodies. Group B represents 67 bank voles thatwere trapped and kept in the laboratory for one month as previouslydescribed (Schoenecker et al., Appl. Anim. Behav. Sci., 68, 349-357,2000). An additional group of 54 animals were examined in Stockholm forinsulin sensitivity and pancreas histology before and after diabetesdevelopment.

The data in Table 1 shows the occurrence of diabetes in the two groupsof bank voles. In the Group A bank voles (n=101), four female animalswere found positive for glucosuria and blood glucose values of 215, 302,313 and 340 mg/dl, respectively, In the remaining bank voles, the meanblood glucose±S.D. was normally distributed at 101±28 mg/dl. The bodyweight of the trapped voles from group A ranged from 8.5-28.4 g, themean value±S.D. was 19±5 g. Occasional hyperglycemic and glucosuric bankvoles may therefore be trapped in the wild. Whether these four animalshad stress-induced hyperglycemia or overt diabetes remains to beestablished. These results in the Group A bank voles differ markedlyfrom the 67 Group B bank voles that were trapped and kept in standardlaboratory mouse cages for one month before they were tested fordiabetes. We observed that 22/67 (33%) of these Group B bank voles had ablood glucose above 200 mg/dl, the range being 211-540 mg/dl. As many as18/22 (82%) had ketones and were polydipsic. Gender differences arecommon in both humans (Harris, Diabetes in America, (ed. Harris, M. I.)(National Institutes of Health, Bethesda, 1995) and in animal models ofdiabetes as well as in captured wild rodents that develop non-insulindependent diabetes when fed laboratory chow (for a review see (Shafriret al., Diabetes Metab. Rev., 8, 179-208, 1992). The bank voles wecaptured were also fed laboratory chow but they were not only glucosuricand hyperglycemic but also positive for ketonuria, ketonemia andhyperlipidemia, all suggestive of type 1 diabetes (data not shown). Aninsulin sensitivity test (Actrapid, Novo Nordisk, Copenhagen, Denmark)was also carried out in 16 randomly selected Stockholm bank voles toexclude diabetes due to insulin resistance. At 60 minutes followinginsulin, four animals with blood glucose levels above 200 mg/dlexperienced 30% decrease in blood glucose, four animals with bloodglucose at 120-200 mg/dl showed 60% decrease while eight animals withblood glucose <120 mg/dl showed a 40% decrease in blood glucose. Thesedata indicate that bank voles with varying blood glucose levels areinsulin sensitive. We therefore next examined the pancreas histology innon-diabetic and diabetic bank voles to test if the classification oftype 1 diabetes was supported by a loss of beta cells.

TABLE 1 The frequency of diabetes in wild caught bank voles and in bankvoles kept in the laboratory. Group of bank voles A. Analyzed B. Trappedand at trap captive N 101 67 M/F ratio 42/59 29/38 Blood glucose (mg/dl)Non-diabetic 101 ± 27  86 ± 24 Diabetic 293 ± 54 346 ± 88 Diabetes n (%)4 (4%) 22 (33%) M/F ratio 0/4 14/8  Mean values ± S.D are shown.Bank Voles Develop Type 1 Diabetes Because of a Specific Loss of BetaCells

The pancreas of all 101 Group A bank voles showed normal islets as didthose of non-diabetic Group B bank voles (FIG. 1). The fourhyperglycemic Group A bank voles had no appreciable islet lesions.Immunostaining with insulin and glucagon antibodies showed a normalislet cell distribution with beta cells located in the center surroundedby glucagon-positive cells (FIGS. 1 a and 1 b). In dramatic contrast,Group B bank voles with diabetes had an almost complete loss ofcentrally located insulin-positive cells that were replaced by prominentvacuolization or fatty infiltration (FIGS. 1 c and 1 d). Islets withinfiltrating mononuclear cells were occasionally observed (FIG. 2) butinsulitis was conspicuously absent in the majority of the bank voles.The beta cell destruction was unique to bank voles with diabetes andindicate that the animal should be classified as having type 1 diabetes.

In order to evaluate whether the Ljungan virus was associated with theislet beta cell lesion we next immunostained the pancreas sections withhigh titer mouse antisera against Ljungan virus (Niklasson et al.,(supra), 1999). We used antisera to two distinct Ljungan virus isolates,87-012 and 145SL and, as controls, eight different antisera preparedwith the same procedure against Rift Valley Fever virus, Ockelbo virus,Langat virus and Sindbis virus. Both the 87-012 and the 145SL Ljunganvirus antisera at dilution of 1:4000 or higher immunostained islets indiabetic but not in non-diabetic bank voles (FIG. 3) visualizing thepresence of Ljungan virus antigen in affected islets. None of thecontrol sera showed immunostaining at a dilution of 1:500 or higher.Furthermore, an analysis of Stockholm bank voles euthanized withvariable blood glucose levels after 2-3 months of captivity revealedthat the severity of beta cell loss was gradual (FIG. 3 panel b, c andd). Also in these apparently early lesions, a mononuclear cellinfiltration was conspicuously absent. Without being bound to any onetheory, it is submitted that the beta cell-specific destruction inassociation with immunoreactive virus antigen suggests that the Ljunganvirus might have had a lytic effect on the beta cells, perhapsaccelerated by the stress of bringing the bank voles into captivity.Although mononuclear cell infiltration was not a prominent feature ofthe beta cell destruction it cannot be excluded that Ljungan virus betacell lysis results in autoantigen presentation that takes place in lymphnodes draining the pancreas or by antigen presenting cells in or aroundthe islets. We therefore next examined the possibility that thedevelopment of diabetes was associated with autoantibodies to the isletautoantigens GAD65, IA-2 or insulin. Autoantibodies to theseautoantigens predict type 1 diabetes in humans (reviewed in Schranz etal., (supra), 1998 and Leslie et al., (supra), 1999) but not in the NODmouse or BB rat models of type 1 diabetes (Bach et al., Endocrine Rev.,15, 516-542, 1994). The possible presence of autoantibodies to theseautoantigens would further support the hypothesis that the bank volesdeveloped type 1 diabetes.

Bank Vole Diabetes is Associated with Autoantibodies to GAD65 and IA-2

Standardized radioligand-binding assays that detect autoantibodies toGAD65 (Grubin et al., (supra), 1994; Hampe et al., (supra), 2000), IA-2(Kawasaki et al., Diabetes, 45, 1344-9, 1996; Vandewalle et al.,(supra), 1997) and insulin (Williams et al., (supra), 1997) were used toanalyze serum samples from available animals of both Group A andCopenhagen group B bank voles (FIG. 4). Compared to Group A animals,GAD65 (P<0.001) but not IA-2 or insulin autoantibodies were increasednon-diabetic Group B bank voles. More importantly, however, diabeticgroup B bank voles had higher GAD65 (P<0.001), IA-2 (P<0.001) andinsulin (P<0.0346) autoantibody levels than the non-diabetic voles (FIG.4). The increased levels of GAD65, IA-2 and insulin autoantibodiesfurther indicates that diabetes in these bank voles should be classifiedas type 1 diabetes.

Since Ljungan virus antigen was demonstrated in the islets of diabeticbank voles (FIG. 3) we next determined whether antibodies to Ljunganvirus antigens were associated to GAD65 and IA-2 autoantibodies. Aradioligand binding assay, similar to the GAD65 and IA-2 autoantibodyassays (Grubin et al., (supra), 1994) was developed with ³⁵S-labelledvirus antigens generated by coupled in vitro transcription andtranslation using the T7 promoter of the Ljungan virus cDNA (unpublishedresults). Although the Group A bank vole sera showed a wide range ofantibody levels against Ljungan virus antigen (FIG. 4), the mean levelsof Ljungan virus antibodies in the non-diabetic Group B bank voles weresignificantly increased (P<0.001). In group B bank voles, the levels ofLjungan virus antibodies were higher in diabetic than non-diabeticanimals (P=0.0015). Since the diabetic Group B bank voles also showedincreased levels of GAD65, IA-2 and insulin autoantibodies, we nexttested if they were related to Ljungan virus antibody levels. In thediabetic Group B bank voles, antibody levels against Ljungan virusantigens correlated with levels of GAD65 (P<0.0001), IA-2 (P<0.0001) andinsulin (P<0.03) autoantibodies. These associations suggest that Ljunganvirus infection may induce an immune response that will also includebeta cell autoantigens. Without being bound to any one theory, thereseems to be two possibilities. The first possibility is that beta celldestruction is leading to autoantigen presentation in draining lymphnodes; the second possibility is that autoantibodies are formed due tomolecular mimicry between virus and the autoantigen. The latterhypothesis was tested by comparing the predicted amino acid sequences ofthe Ljungan virus cDNA with those of GAD65, IA-2 and insulin.

Ljungan Virus Molecular Mimicry to Islet Autoantigens

The comparison between the Ljungan virus amino acid sequence predictedfrom the cDNA and the GAD65, IA-2 and insulin sequences revealed severalpotential regions of sequence similarities (FIG. 5 a). We searched 1514picornavirus proteins in Genbank's viral taxonomy at NCBI usingstand-alone BLAST and found that these homologies were exclusively foundin parechoviruses (echovirus 22/23) isolates (data not shown). Theregions indicated for GAD65, 237-241 and 449-452 have been implicated inthe middle and C-terminal GAD65 autoantibody binding sites (Schwartz etal., J. Mol. Biol., 287, 983-999,1999). While the 561-569 is outside,the 964-976 sequence is within reported autoantibody binding sites fortype 1 diabetes associated IA-2 autoantibodies (Leslie et al., (supra),1999). The most interesting significant relationship was between theLjungan virus antigen and the 45-54 insulin since the 45-54 homologymaps to the insulin active site (Steiner et al., Diab. Care, 13,600-609,1990). Since antibody levels against Ljungan virus in vitrotranslated antigens correlated to levels of both GAD65 and IA-2autoantibodies in the diabetic Group B bank voles and because of thesignificant sequence similarities (FIG. 5 a), we next tested whetherLjungan virus antisera would immunoprecipitate any of the isletautoantigens. While labeled mouse GAD65 was not recognized, the mouseLjungan virus polyclonal antiserum 87-012 (Niklasson et al., (supra),1999) was capable of immunoprecipitating human GAD65 (FIG. 5 b),indicating significant epitope specificity (Hampe et al., (supra),2000). The Ljungan virus in vitro translated antigens immunoprecipitatedby the 87-012 Ljungan virus antiserum was reciprocally displaced by bothcold Ljungan virus in vitro translated antigens and human GAD65 but notby human proinsulin (FIG. 5 c). The human type 1 diabetes GAD65antibody-positive serum #591 showed concentration dependentimmunoprecipitation of Ljungan virus in vitro translated antigens andboth human and mouse GAD65 (FIG. 5 d). Cold Ljungan virus in vitrotranslated antigens but not GAD65 nor proinsulin, displaced theimmunoprecipitation of Ljungan virus in vitro translated antigens by thehuman serum (FIG. 5 e). These observations support the possibility ofantibody cross-reactivity due to molecular mimicry between Ljungan virusantigen and GAD65 in mice inoculated by Ljungan virus.

Example 2

Individuals Infected with Ljungan Virus are Susceptible to DevelopingDiabetes

We tested to see if new onset type 1 diabetes children had Ljungan virusantibodies by both standard immunofluorescence and radioligand bindingassay with Ljungan virus cDNA in vitro translated antigens.

Children with Type 1 Diabetes have Ljungan Virus Antibodies

The commonly used indirect immunofluorescence virus antibody test wasfirst compared to the radioligand binding assay for Ljungan virusantigen antibodies (FIG. 6). There was a significant correlation(Spearman Rank Sum correlation) between the two assays (P<0.001).Compared to the 17 healthy control children, the children with new onsetType 1 diabetes had increased levels of Ljungan virus antibodies scoredin the immunofluorescence assay (P<0.00l) (FIG. 6). These data indicatethat children with new onset diabetes may have been exposed to Ljunganvirus.

Discussion

The examples provide evidence that wild caught bank voles may developtype 1 diabetes associated with specific beta-cell destruction,insulitis and autoantibodies to GAD65 and IA-2. Our observation that itwas possible to detect Ljungan virus antigen in affected pancreaticislets showing gradual destruction and end-stage fatty degeneration alsoindicates that this virus causes or at least contributes to the loss ofbeta cells. In addition, the diabetic bank voles had increased levels ofantibodies to Ljungan virus cDNA in vitro translated virus antigens. Thelevels of these antibodies were also found to correlate to the levels ofautoantibodies to both GAD65 and IA-2. These data indicate that diabetesobserved in both captured bank voles and in bank voles born to captiveanimals represents type 1 diabetes. Although the histology of the isletsin diabetic voles may be consistent with an acute lytic effect andpresence of viral antigen, it cannot be excluded that beta cells mayalso have been lost by an ensuing T cell or antibody-mediated cellulartoxicity. Also consistent with type 1 diabetes were our observationsthat islet beta cells were specifically lost. The prominentvacuolization or fatty infiltration seems unique to the bank volesdiabetes and differ from other virus causing diabetes in rodents whereinsulitis is seen more often. In particular, the rare occurrence ofislets infiltrated with mononuclear cells suggest that the bank voleislet lesion is less associated with insulitis compared to otherdiabetogenic virus (Jun et al., (supra), 2001; Rayfield et al.,Diabetes, 27, 1126-1140, 1978; Vaaralae et al., (supra) , 1999).

Diabetes in bank voles was first described during a study of stereotypicbehavior in bank voles (Schoenecker et al., Appl. Anim. Behav. Sci., 68,349-357, 2000) When captured in the wild, brought to the laboratory tobe kept in standard laboratory mouse cages, and fed laboratory chow,bank voles developed polydipsia and glucosuria. Diabetes was detected in4/101 Group A animals that were euthanized in the forest at the site ofthe trap which was different from the 22/67 bank voles kept in thelaboratory. Our data suggest that the diabetes symptoms in these animalsfulfill current classification criteria for autoimmune type 1 diabetesin humans (Mellitus et al., Diabetes Care, 20, 1183-1197, 1997). Thediabetic bank voles sustain hyperglycemia, ketonemia, ketonuria,hyperlipidemia and weight loss, all criteria that are consistent withtype 1 diabetes classification. In addition, the diabetic bank voles hadincreased levels of both GAD65, IA-2 and insulin autoantibodies. Theseautoantibodies predict type 1 diabetes in humans (Verge et al.,Diabetes, 45, 926-933, 1996); Bingley et al., Diabetes Care, 22,1796-801, 1999) and confirm the type 1 diabetes classification.

The short period of time it took for bank voles to develop diabetesafter capture, and the complete islet beta cell destruction in diabeticvoles associated with positive immunostaining for Ljungan virus antigensuggest that acceleration of an existing, low-level viral infection mayinduce disease. While the exact mechanism of such a process remainsunclear, our initial studies of diabetic bank voles indicate that stressis involved in diabetes development. Early experimental stress may leadto increases in adult adrenocortical stress responses (King et al.,Horm. Behav., 36, 79-85, 1999) and such stress responses may act as astimulus of virus replication in beta cells. This speculation issupported by the observation that stress induced by swimming increasedthe frequency of diabetes in our wild caught bank voles (data notshown). Stress has also been implicated in human type 1 diabetes sincenegative life events increased the risk for childhood type 1 diabetes(Thernlund et al., Psychological Stress and the Onset of IDDM inchildren, 18, 1995; Hagglof et al., Diabetologia, 34, 579-83, 1991;Dahlquist et al., Diabetologia, 34, 757-762, 1991). Similarrelationships could be relevant to our bank voles and therefore aid inunderstanding the etiology of diabetes.

It is now possible to identify other mechanisms that induce Ljunganvirus-associated diabetes in bank voles and to conduct studies onintervention and protection, for example by anti-viral agents orreducing responses to stress.

It has also been found that very high Ljungan virus antigen antibodylevels are observed in some of the non-diabetic Group A bank voles. Thismay reflect a neutralizing and protective Ljungan virus immune responsewith implications for future vaccination approaches.

Taken together we have demonstrated, first, that bank voles developdiabetes that fulfills the criteria for type 1 diabetes: diabeticanimals showed persistent hyperglycemia associated with weight loss,ketosis and hyperlipidemia (data not shown) as well as insulindependence associated with specific beta-cell destruction and insulitis.Second, diabetic voles had increased levels of autoantibodies to GAD65and IA-2, and that these autoantibodies correlated to Ljungan virusantigen antibodies. Third, the association between Ljungan virus andbank vole diabetes was supported by the presence of Ljungan virusantigen detected by irnmunocytochemistry in affected diabetic bank voleislets. Fourth, there was significant molecular mimicry between theLjungan virus polyprotein and GAD65, IA-2, and insulin isletautoantigens, illustrated by GAD65 cross reactivity of high titer mouseand guinea-pig Ljungan virus antisera. Finally, a relationship betweenLjungan virus infection and human type 1 diabetes was indicated byincreased levels of Ljungan virus antibodies in children with newlydiagnosed type 1 diabetes.

Example 3

Data on Diabetes Mellitus in Other Mammals than Bank Voles

1. Field Voles (Microtus agrestis)

The field vole develops clinical diabetes with symptoms of polydipsiaand polyuria identical to bank voles. Ljungan virus has been isolatedfrom field voles with type 1 diabetes trapped in Sweden. It is likelythat field voles are just as good an animal model for type 1 diabetes asthe bank vole.

2. Cats

Between 1 in 50-500 will develop the disease in a lifetime. The clinicaldisease mimics in some animals type 1 diabetes and in some animals type2 diabetes. Amyloid deposits localized to the islets of Langerhans aretypical of type 2 diabetes mellitus. However, diabetic cats mostcommonly have pancreatic islet destruction associated with pancreaticamyloidosis and are insulin deficient like type 1 diabetes. The diseaseoccurs in all ages of the cats but the majority of diabetes affect oldercats (Westermark et al., PNAS USA, 84, 3881-5, 1987; Johnson et al.,Veterinary Pathology, 22(5) :463-8, 1985; and Yano et al., VeterinaryPathology, 18(3) : 310-5, 1981). We have investigated the pancreastissue of diabetic type 1 and type 2 as well as normal cats usingimmunohistochemistry (IHC). As in the bank voles Ljungan virus could bedetected in the destroyed islets of the pancreas in the diabetic type 1and 2 cats but not in the normal cats (Bo Niklasson unpublishedobservations). This is very strong evidence suggesting that Ljunganvirus cause diabetes in cats.

3. Dogs

Between 1 in 50-500 will develop the disease in a lifetime. A review ofthe literature has shown that over half of the documented diabetic dogsare type I diabetes. However, type 2 is also common in dogs with obesity(Stogdale L. Cornell Veterinarian. 76(2) :156-74, 1986). We haveinvestigated the pancreas tissue of diabetic type 1 and type 2 as wellas normal cats using immunohistochemisty (IHC). As in the bank volesLjungan virus could be detected in the destroyed islets of the pancreasin the diabetic type 1 and 2 dogs but not in the normal dogs (BoNiklasson unpublished observation). This is very strong evidencesuggesting that Ljungan virus caused the disease.

4. Guinea Pigs

Spontaneous diabetes mellitus in guinea pigs, parallels in many ways thesyndrome known as juvenile diabetes mellitus in man: elevated bloodglucose levels; reproductive dysfunction in the female; degranulationand severe cytoplasmic vacuolation of beta cells, severe fattydegeneration of acinar cells, and hyperplasia of the islets of thepancreas; and a high frequency of abnormal pancreatic secretions (Langet al., Diabetes, 25(5) :434-43, 1976). The severity of pathologicchanges in the pancreatic islets parallel, in general, the severity ofthe clinical symptoms. The other clinical parameters of note areelevated serum triglycerides, normal serum but elevated aorticcholesterol, and absence of ketonemia or ketonuria. Microangiopathy,another characteristic of juvenile diabetes mellitus in man wasdemonstrated as a significant increase in the thickness of the basalmembranes in peripheral capillaries. A glomerular lesion encountered insome of the diabetic guinea pigs was shown to be similar to theglomerular sclerosis seen in human diabetics. Although a definitiveetiologic agent was not identified, the disease was clearly contagiousin origin.

5. Rabbits

Spontaneous diabetes mellitus has been observed in a female New Zealandwhite rabbit. Three groups of animals could be identified. Some animalshad overt diabetes characterized by fasting hyperglycemia and depressedintravenous glucose stimulated serum insulin levels (Conaway et al.,Clinical & Experimental, 30(1) :50-6, 1981). This abnormality is seenbetween 1 and 3 years of life. Another group of animals developedabnormal glucose disposal with normal or slight elevations in fastingserum glucose levels. Glucose stimulated insulin levels are alsosignificantly lower in the rabbits with abnormal glucose disposal. Theremaining animals exhibit no apparent abnormalities of glucosemetabolism. Despite marked increases in serum and urinary glucose, onlymild ketonemia was observed. The relatively late onset of diabeticsymptoms, lack of obesity, severe hyperglycemia, and depressed insulinsecretion without ketoacidosis make this a model with many of thecharacteristics of insulin responsive diabetes as seen in non-obesehuman adults.

6. Hamster

Chinese hamsters spontaneously develop diabetes mellitus andcardiomyopathy. The diabetic hamsters, shows body weight loss,hyperglycemia (mean fasting plasma glucose 402 mg/dl), hypoinsulinemia,hyperlipidemia and ketonemia. The diabetic hamsters showed reducedactivities of cytoplasmic glycolytic key enzymes: hexokinase, pyruvatekinase and phosphofructokinase; increases in cardiac glycogen andglucose-6-phosphate contents; and a 40% decrease in cardiac ATP content,indicating decreased energy production. An accumulation of myocardialtriglyceride and cholesterol was found in the diabetic hamsters (Eto etal., Diabetes Research & Clinical Practice, 3(6) :297-305, 1987).

Syrian hamsters infected with rubella virus passaged in beta-cells alsodevelops diabetes that closely parallels the diabetes observed withcongenital rubella (Rayfield et al., Diabetes, 35(11) : 1278-81, 1986).The hamsters develop hyperglycemia and hypoinsulinemia, which aresustained throughout the 15 week study period. A mononuclearinfiltration of the islets, isolation of rubella virus from wholepancreas, the presence of viral antigen in beta-cells byimmunofluorescence localization, and cytoplasmic islet cell antibodies(40%) are demonstrated. These data suggest that an autoimmune processand diabetes develop after rubella virus infection in neonatal hamsters.This model may uncover the precise mechanism by which rubella virusinduces similar disease in humans.

All documents referred to in the above description are incorporatedherein by reference.

1. A method for obtaining an animal model for human diabetes, comprisingobtaining a mammal; determining that the mammal is infected with aLjungan virus; modulating the immune system of the infected mammal tofacilitate the development of diabetes, wherein the immune system ismodulated by subjecting the infected mammal to stress; and obtaining ananimal model for human diabetes, wherein the infected mammal developsdiabetes.
 2. The method according to claim 1, wherein the methodadditionally comprises determining whether the mammal has signs ofketoacidosis and high blood glucose levels that can be reduced byinsulin.
 3. The method according to claim 1, wherein the methodadditionally comprises testing for the presence of autoantibodies to atleast one of GAD65, IA-2 and insulin.
 4. The method according to claim1, wherein the mammal is subjected to stress for at least about 2months.
 5. The method according to claim 4, wherein the mammal issubjected to stress by keeping it in a cage.
 6. The method according toclaim 1, wherein the mammal is a rodent.
 7. The method according toclaim 1, wherein the mammal is a bank vole.
 8. The method according toclaim 7, wherein the bank vole is obtained from the wild or is theprogeny of a bank vole obtained from the wild.
 9. The method accordingto claim 8, wherein the bank vole is obtained from Denmark, Sweden orFinland.